Colloidal solutions or sols can be formed by condensation processes. Conventionally, a reducing agent is added to a metal salt forming metallic particles.
The most frequently employed method for preparing gold hydrosols is the reduction of chloroauric acid with a suitable reducing agent. A number of reducing agents can be used including formaldehyde, hydrogen peroxide, phosphorus, substituted ammonias such as hydroxylamine and hydrazine. Methods of production of gold sols are generally discussed in Roth, J. "The Colloidal Gold Marker System for Light and Electron Microscopic Cytochemistry", In Bullock, G. R. and Petrusz, P. (eds.), Techniques in Immunocytochemistry 2, pp. 217-284; Horisberger, M. "Colloidal Gold: A Cytochemical Marker For Light and Fluorescent Microscopy and For Transmission and Scanning Electron Microscopy", SEM, 11:9-31 (1981); Weiser, H. B., "The Colloidal Elements", in Inorganic Colloid Chemistry, J. Wiley & Sons (1933), pp. 1-107.
Gold particles ranging in size from 2-140 nm are readily produced by a variety of methods. See Geoghegon, W. D. "Immunoassays at the Microscopic Level: Solid-Phase Colloidal Gold Methods", J. Clin Immunoassay 11(1):11-23 (1988). Using trisodium citrate as a reductant, particle diameter has been found to be a function of the quantity of citrate added. See Frens, G. "Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions", Nature Physical Sci. 241:20-22 (1973). Particle size may be varied over an approximate size range of 15-140 nm by varying the amount of citrate added. Particles in the 2-20 nm range can be produced using white phosphorus, ascorbic acid, or a mixture of tannic acid and citrate as the reductant.
Colloidal gold can be used as a particulate marker for the detection and localization of target molecules by various modes of microscopy using both direct and indirect labelling approaches. Under appropriate conditions, colloidal gold will bind macromolecules by non-covalent electrostatic adsorption with little change in the specific activity of the bound macromolecule. Interaction is influenced by ionic concentration, pH conditions in correlation with protein isoelectric points and protein stabilizing levels.
Under appropriate conditions, metal sol particles can be labelled with a variety of macromolecules, including polysaccharides, glycoproteins, proteins, lectins and antibodies.
Whenever the term "metal sol particles" is used in this application, this is understood to mean particles of a sol consisting of a metal or transition metal, a metal or transition metal compound or polymer nuclei coated with a metal or transition metal or a metal or transition metal compound. The metal sols may be of metals or transition metals or compounds thereof such as oxides, hydroxides, and salts or, of polymer nuclei coated with metals or transition metals or compounds thereof. Examples include platinum, gold, silver, iron, copper, selenium, chromium, vanadium, titanium, manganese. In addition, it is recognized that a metal sol produced in accordance with the teachings of Applicants' invention may be converted to a suspension of insoluble metal salts, sulphides, oxides, hydroxides or similar compounds. In general, all metals, transition metals or compounds thereof, which may be readily demonstrated by means of techniques well known in the art are within the scope of Applicants' invention.
For a general discussion concerning gold particle labelling techniques, see Horisberger, M. "Colloidal Gold: A Cytochemical Marker For Light and Fluorescent Microscopy and For Transmission and Scanning Electron Microscopy", SEM 11:9-31 (1981). In most cases there is little change in the bioactivity of the adsorbed molecules. Generally, these probes acquire the specific activity of the adsorbed macromolecule and their stability upon storage is good. However, when gold particles are labelled with proteins, full stabilization against coagulation by electrolytes is not always observed, especially with larger size markers. A number of stabilizing agents including polyvinylpyrrolidone, poly-L-lysine, poly-L-proline, polyethylene glycols (PEG), and Carbowax have been suggested.
As probes, gold particles are particularly interesting because their electron dense properties allow detection by transmission electron microscopy (TEM), their capability of strong emission of secondary electrons allows visualization by scanning electron microscopy (SEM), their characteristic X-ray signals allow identification of gold markers on cell surfaces. In addition, gold probes are also useful in fluorescent microscopy by labelling gold particles with fluorescent molecules. Gold particles bound to a cell surface appear as an orange-red coating and are therefore useful in photonic microscopy and in macroscopic observations. The advantages of gold probes are discussed generally in Horisberger, M. "Colloidal Gold: A Cytochemical Marker For Light and Fluorescent Microscopy and For Transmission and Scanning Electron Microscopy", SEM, 11:9-31 (1981); Goodman, S. L. et al. "A Review of the Colloidal Gold Marker System", SEM 11:133-146 (1980). A bibliography of gold probe labelling studies is provided by Goodman, at page 139.
It has been shown that it is possible to select and adsorb a specific substance to colloidal gold to optimize its use as a tracer for electron microscopy (EM). It has been suggested that an ideal tracer substance should be available in a wide range of uniform sizes with an electron scattering core surrounded by a coat that could be varied as needed. See Geoghegon, W. D. "Immunoassays at the Microscopic Level: Solid-Phase Colloidal Gold Methods", J. Clin Immunoassay 11(1):11-23 (1988). The gold is stabilized by a variety of substances, including polypeptides, polynucleotides, polysaccharides and organic polymers.
There are a number of immunoassays employing colloidal gold. The presence of a reactive protein on a probe can be demonstrated and quantitated by direct and indirect radioactive binding assays and agglutination assays. See Goodman, S. L. et al. "A Review of the Colloidal Gold Marker System", SEM 11:133-146 (1980).
To make a gold probe, the following basic steps are followed: a protein solution and colloidal gold are pH adjusted to optimize protein adsorption, the minimal protecting amount of protein is determined, the appropriate amount of protein is mixed with the colloidal gold and a secondary stabilizer added. The gold probe can then be purified and its concentration adjusted to a predetermined optical density. Using these general principles, ligands can be bound to gold probes for use in immunoassays. The Janssen manual entitled "Colloidal Gold Sols for Macromolecular Labelling" (1985) discloses basic probe construction techniques.
U.S. Pat. No. 4,313,734 (Leuvering) discloses a metal sol particle immunoassay. Leuvering teaches the preparation of a gold sol by reducing chloroauric acid with trisodium citrate to produce gold particles with diameters of 45-70 nm. The particles are then labelled with a rabbit anti-HPL serum by adjusting the sol to pH 7.0 with potassium carbonate and then adding a rabbit anti-HPL immunoglobulin solution. The coated gold particles can then be used for determination of HPL by colorimetry or atomic adsorption spectrophotometry. Leuvering also discloses the visual detection of hepatitis B surface antigen (HBsAg) by means of a gold particle sheep anti-HBsAg immunoglobulin conjugate. In this method the gold sol and the gold-immunoglobulin conjugate are prepared as before with the exception that a diluted solution of the sheep anti-HBsAg immunoglobulin solution was used instead of the rabbit anti-HPL immunoglobulin solution. Leuvering also discloses the determination of HCG with the aid of a gold particle-anti-HCG conjugate in an agglutination test. The gold sol and gold particle anti-HCG conjugate are prepared using the previously described methods. A competitive receptor assay for HCG is also disclosed. A gold dispersion consisting of particles having a diameter between 6-15 nm is prepared by adding sodium citrate to a boiling solution of chloroauric acid. A dialyzed HCG solution was added to the gold dispersion. The mixture was stabilized with Carbowax. A sandwich assay for HCG using an insolubilized HCG receptor and the gold particle-anti HCG conjugate is then performed.
U.S. Pat. No. 4,775,636 (Moeremans et al.) discloses a blot overlay assay using colloidal metal particles, preferably 3-100 nm in size. Moeremans et al. teach the use of a dispersion of a metal or metal compound or nuclei coated with a metal or metal compound as one detection principle in blot overlay techniques. Colloidal metal particles, including gold particles are prepared following art-known procedures. The colloidal metal particles can then be attached directly or indirectly to the specific binding agent or to a macromolecule that binds specifically to the first specific binding agent using known art procedures. Moeremans et al. find that colloidal metal particles so prepared will accumulate at the specific binding sites and surprisingly become visible as the color characteristic for the colloidal metal particles used, e.g. from pink to a dark red color, in the case of gold. The signal is read by eye or using spectrophotometric procedures.